The invention relates to the field of analytical detection of psychoactive drugs of the phenethylamine family. Specifically, immunodetection of 2C and DO phenethylamines, sub-families of the phenethylamines, is described. The psychoactive phenethylamines represent a large family of drugs, each member incorporating the phenethylamine sub-structure (FIG. 1) and are exemplified by the legal, but abused, therapeutic drugs amphetamine and methamphetamine, the plant-derived compounds cathinone and mescaline (3,4,5-trimethoxyphenethylmine) and the designer drugs MDMA and mephedrone. The large number of phenethylamines can be approximately classified to sub-families according to the nature of the chemical substituents on the basic phenethylamine sub-structure;—                the methylenedioxy-phenethylamines represented by MDMA,        the β-keto-phenethylamines represented by cathinone/mephedrone/methcathinone, including pyrrolidinophenones, typically classified as a sub-set of the synthetic cathinones, such as RS)-1-(benzo[d][1,3]dioxol-5-yl)-2-(pyrrolidin-1-yl)pentan-1-one (commonly referred to as 3,4-methylenedioxypyrovalerone or MDPV (see EP Patent publication 2626358) and        the 2,5-dimethoxy-phenethylamines represented by, for example, 1-(2,5-dimethoxy-4-iodophenyl)ethan-2-amine (2C-I) and 1-(4-bromo-2,5-dimethoxyphenyl)propan-2-amine (DOB) (FIG. 1).        
Detection of the psychoactive phenethylamines is a necessary component in the clinical and forensic toxicological fields and is finding increasing use in work-place testing to support health and safety. The main analytical technique used for identifying individual psychoactive phenethylamines is mass spectrometry (MS). This expensive and specialised confirmatory technique is generally preceded by the immunoassay, a cheaper and simpler technique using antibodies with both generic and molecule-specific detection capabilities.
There are currently immunoassay based methods for several sub-families of the phenethylamine class of psychoactive drugs, including mephedrone/cathinone (European Patent No. 2442110), the pyrrolidinophenones, typically classified as a sub-set of the synthetic cathinones (see EP Patent publication 2626358), the amphetamines and MDMA (European Patent No. 1321772). Depending on the application, either generic or molecule-specific antibodies may be desirable. For a comprehensive pre-screen where there are tens of molecules in a family, generic antibodies might be preferred with MS used subsequently to identify an individual molecule. Alternatively, where the requirement might be to identify a sub-family of molecules; an immunoassay specific to the sub-family could preclude, or better support, subsequent analysis by MS.
Swortwood et al 2013, at Table 7, examines the cross-reactivity for a Randox MDPV kit (all tested members of the DO and 2C sub-families show less than 0.1% cross-reactivity. Specifically, of the DO sub-family, each of tested (±)-DOET, (±)-DOM and (±)-DOB show less than 0.1% cross-reactivity when compared to 100% for (±)-MDPV and, of the 2C sub-family, each of tested 2C-E, 2C-B and 2C-T-7 show less than 0.1% cross-reactivity when compared to 100% for (±)-MDPV. Swortwood et al 2013, at Table 7, also examines the cross-reactivity for a Randox mephedrone/methcathinone kit (all tested members of the DO and 2C sub-families show less than 0.0125% cross-reactivity). Specifically, of the DO sub-family, each of tested (±)-DOET, (±)-DOM and (±)-DOB show less than 0.0125% cross-reactivity when compared to 100% for (±)-mephedrone and, of the 2C sub-family, each of tested 2C-E, 2C-B and 2C-T-7 show less than 0.0125% cross-reactivity when compared to 100% for (±)-mephedrone. Swortwood et al 2013 concludes that there are no current phenethylamine immunoassays dedicated to the detection of, separately, members of the 2C and DO sub-families.
Petrie et a12013, at Table 1, reviews cross-reactivity of “bath salts” using commercially available amphetamine screening immunoassays—AxSYM amphetamine/methamphetamine II assay (Abbott); CEDIA amphetamine/ecstasy immunoassay (Thermo Fisher); and EMIT II Plus amphetamines assay (Siemens). The AxSYM assay does not show cross-reactivity to any tested member of either the DO sub-family (DOB, DOEt and DOM) or the 2C sub-family (2C-I, 2C-B, 2C-T-2, 2C-T-4, 2C-T-7 and 2C-H), even when tested at 5,000 ng/mL. The EMIT and CEDIA amphetamine assays have been shown to weakly bind to both 2C and DO sub-family members when tested at concentrations of 5,000 ng/ml (Petrie et al 2013). The CEDIA assay shows cross-reactivities of none (2C-I), 3.0% (2C-B) 2.2% (2C-T-2), 2.2% (2C-T-4), 2.8% (2C-T-7) and 3.1% (2C-H) for the 2C sub-family and shows very slightly higher cross-reactivities of 8.5% (DOB), 4.8% (DOEt) and 3.9% (DOM) for the DO sub-family. The EMIT assay shows cross-reactivities of 28.6% (2C-I), 36.0% (2C-B), 11.7% (2C-T-2), none (2C-T-4), 18.1 (2C-T-7) and none (2C-H) for the 2C sub-family and shows very slightly higher cross-reactivities of 39.1% (DOB), 18.8% (DOEt) and 23.2% (DOM) for the DO sub-family, albeit at very high and unrealistic concentrations. Each of the AxSYM, CEDIA and EMIT II Plus assays were tested at 5,000, 20,000 and 100,000 ng/mL. Petrie et al 2013 admits a urinary level of 100,000 ng/mL would likely only be encountered in severe overdose cases. None of the AxSYM, CEDIA and EMIT II Plus assays can be used to discriminate between drugs of the DO and 2C sub-families. This lack of sensitivity and specificity to a single sub-family (either 2C or DO) precludes the CEDIA and EMIT assays from being practically applied to detect, and discriminate between 2C and DO drugs. The inability to even bind to tested members of the DO and 2C sub-families precludes the use of AxSYM assay as a screen for members of either the DO and 2C sub-families.
A further problem facing immunodetection of phenethylamines is the potential for antibodies to exhibit unexpected and undesirable cross-reactivity to common nutritional supplements and commonly prescribed medicines giving rise to false positive assay results. For example, the anorectic dimethylamylamine has been shown to cross-react with amphetamine immunoassays (Vorce et al 2011).
Thus, there is a need for the sensitive and selective immunodetection of 2C and DO drugs, while avoiding unwanted cross-reactivity.